This invention concerns Factor IX in general, and particularly concerns Factor IX containing a mutation that enhances the clotting activity thereof. This invention also concerns DNA constructs encoding such Factor IX, along with vectors containing such constructs.
Factor IX (FIX; also known as xe2x80x9cChristmas Factorxe2x80x9d) plays a key role in both the intrinsic and extrinsic coagulation pathways (E. Davie et al., Biochemistry 30, 10363 (1991); B. Furie and B. Furie, Cell 53, 505 (1988)). Human Factor IX and DNA encoding the same is disclosed in U.S. Pat. No. 4,994,371 to Davie et al., and in European Patent 0107278 to Brownlee.
FIX circulates as a 415 amino acid, single chain plasma zymogen (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993)). The zymogen of FIX is activated by FXIa or by the tissue factor/FVIIa complex. Specific cleavages between arginine-alanine 145-146 and arginine-valine 180-181 result in a light chain and a heavy chain linked by a single disulfide bond between cysteine 132 and cysteine 289 (S. Bajaj et al., Biochemistry 22, 4047 (1983)). The structural organization of FIX is similar to that of the vitamin K-dependent blood clotting proteins FVII, FX and protein C (B. Furie and B. Furie, supra). The approximately 45 amino acids of the amino terminus comprise the gamma-carboxyglutamic acid, or gla, domain. This is followed by two epidermal growth factor homology domains (EGF), an activation peptide and the catalytic xe2x80x9cheavy chainxe2x80x9d which is a member of the serine protease family (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993); S. Spitzer et al., Biochemical Journal 265, 219 (1990); H. Brandstetter et al., Proc. Natl. Acad Sci. USA 92, 9796 (1995)).
The major physiological function of FIXa in the blood coagulation cascade is to convert FX to FXa in a process that requires a phospholipid surface, calcium ions and FIXa""s protein cofactor, FVIIIa. FIXa alone is an extremely poor protease but when bound to FVIIIa to form the xe2x80x9ctenasexe2x80x9d complex it becomes a potent FX activator(E. Duffy et al., J. Biol. Chem. 267, 17006 (1992); G. van Dieijen et al., J. Biol. Chem. 256, 3433 (1981)). It has been proposed that, upon binding FVIIIa, FIXa may undergo a conformational change at or near the active site (V. Mutucumarana et al., J. Biol. Chem. 267, 17012 (1992)). The importance of FIX is exemplified by the fact that patients with defective FIX molecules suffer from hemophilia B, an X-linked, recessive bleeding disorder which is clinically indistinguishable from hemophilia A in patients who have no FVIII function.
A first aspect of the present invention is a Factor IX protein having an amino acid substitution at amino acid position 338. Factor IX of the present invention is non-naturally occuring (e.g., does not contain only an arginine to proline substitution at amino acid position 338). The FIX proteins of the present invention advantageously have increased clotting activity as compared to the corresponding wild-type molecule.
A second aspect of the present invention is a pharmaceutical formulation comprising a mammalian Factor IX as described above in combination with a pharmaceutically acceptable carrier.
A third aspect of the present invention is a method of facilitating blood clotting in a subject in need of such treatment, comprising administering to the subject a mammalian Factor IX protein as described above, in an amount sufficient to facilitate or enhance blood clotting in said patient.
A forth aspect of the present invention is an isolated nucleic acid (e.g., a DNA or an RNA) encoding a mammalian Factor IX protein as described above.
A fifth aspect of the present invention is an expression cassette containing a nucleic acid encoding a mammalian Factor IX protein as described above.
A sixth aspect of the present invention is a gene transfer vector containing an expression cassette as described above.
An illustrative nucleic acid of the present invention is provided herein as SEQ ID NO:1, and an illustrative Factor IX amino acid sequence of the present invention is provided herein as SEQ ID NO:2.
The foregoing and other objects and aspects of the present invention are explained in greater detail in the drawings herein and the specification set forth below.